Pain disorders in the trigeminal system are reported frequently by millions of Americans. Although pain does not discriminate between men and women, epidemiological studies have demonstrated that women are at increased risk for trigeminal and other forms of pain. Women are more likely to report a variety of temporary and persistent pains and to report more severe pain, more frequent pain and pain of longer duration than men. Importantly, under controlled experimental conditions, women are generally more sensitive to pain-causing stimuli than men. Our preliminary studies identified estrogen receptor 1 (ER1) in rat trigeminal ganglia (TG) neurons and showed that estradiol rapidly (15 min) enhances cellular signaling by two inflammatory mediators, bradykinin (BK) and prostaglandin E2 (PGE2) in cultured TG neurons. Our overall hypothesis is that estrogens, acting via rapid signaling pathways, increase the responsiveness of TG nociceptors to stimulation by inflammatory mediators and that this plays a role in the enhanced pain sensitivity of women. Specific Aim 1: Characterize rapid effects of estradiol (17_-E2) on BK and PGE2 signaling in trigeminal ganglion. We will determine the concentration- and time- and ligand-dependence of 17_-E2 action and identify the estrogen receptor that mediates the rapid action using siRNA knock-down. Specific Aim 2: Identify the rapid 17_-E2-induced changes in the BK and PGE2 receptor systems that are responsible for enhancement of signaling in TG cultures. We will determine if the mechanism for the increased signaling efficacy of BK and PGE2 is due to increases in cell-surface receptor density or to changes in signal transduction efficiency (receptor-G protein coupling efficiency or trafficking to/from membrane microdomains (lipid rafts). Specific Aim 3: Determine the effect of 17_-E2 on nociception in an orofacial model of acute inflammation in female rats. We will determine if exogenously applied 17_-E2 in vivo alters nocifensive behavioral responses to BK, PGE2 or to formalin. In addition, we will assess the role of endogenous 17_-E2 during the estrus cycle on orofacial nociception. These integrated studies will increase our understanding of the consequences and the mechanisms by which estrogen, acting via rapid signaling mechanisms, regulates signaling and nociception by the inflammatory mediators BK and PGE2 in the rat trigeminal ganglion. Detailed study of estrogen receptors and their effects on inflammatory mediator signaling in TG will provide a basis for understanding the enhanced sensitivity of women to pain and provide new targets for improved pain pharmacotherapy. Project Narrative Although pain does not discriminate between men and women, epidemiological studies have demonstrated that women are at increased risk for orofacial and other forms of pain. The integrated studies proposed here will increase our understanding of the consequences and the mechanisms of the female sex hormone, estrogen, that regulate responsiveness of sensory neurons to painful stimuli. This work will provide a basis for understanding the enhanced sensitivity of women to pain and provide new targets for improved pain pharmacotherapy.